Internal pipette solution

Pipette exactly lOmL of this sample stock and lOmL of internal standard solution into a 200-mL volumetric flask and dilute to volume with mobile phase. 

Prepare a 500 ppm analyte standard solution as follows pipette 1 ml of stock solution and 1 ml of the internal standard solution into a 50 ml volumetric flask then dilute to volume with 40% (v/v) ethanol/water. Prepare sample solutions by diluting 1 ml of internal standard to 50 ml with proprietary whisky. Make duplicate 0.5 pi injections of the standard solution and of all sample solutions. From the peak area data of the standard solutions calculate the detector response factors for each component relative to n-butanol. The parts per million (% v/v) amounts of each component in the sample(s) can be determined. As an alternative to capillary GC the analysis can be carried out on the following packed column—15% PEG400 on supasorb 60-80 (2M). 

Internal standard solution (IS) Pipette 100 [iL of n-hexadecane and diluted with ethyl... 

Another important aspect of the holder is its cleanliness. Sometimes the pipette solutions can dirty the holder, forming a thin film of salt in its internal surface, which consists in an important noise source. Holders have to be soaked with methanol and dried with dry clean air or nitrogen before use, and each time that a contamination with salt solutions is suspected. 

As the direct communication is established between the pipette and the intracellular compartment in whole-cell configuration, the intracellular solution is rapidly dialyzed by the pipette solution, giving a control of the internal composition of the cell [29]. Nevertheless, as during the cell dialysis the intracellular material is extensively diluted this can be reflected by a run-down of the activity of some channels, whose normal function depends on the presence of some intracellular components, as for example, the calcium channel. The membrane potential of the cell in the whole-cell configuration is the same as that applied to the pipette, and negative currents are interpreted as cationic currents flowing into the cell, while positive currents represent outward cationic currents. 

Outside-out configuration From the whole-cell configuration it is possible to achieve a new excised-patch. Withdrawing the patch pipette very slowly (Fig. 9A), a small membrane vesicle can be formed on the tip of the pipette (Fig. 9B). This is the outside-out configuration, where the outer surface of the membrane is exposed to the bath solution, and the internal surface of the patch to the pipette solution. In this configuration the pipette potential is equal to that of the membrane, and the recorded current convention is the same as that in whole-cell configuration. 

Another parameter essential for quantitative applications of micropipettes is the internal ohmic resistance, R. It is largely determined by the solution resistance inside the narrow shaft of the pipette, and can be minimized by producing short (patch-type) pipettes. The micropipette resistance has been evaluated from AC impedance measurements. Beattie et al. measured the resistance of micropipettes filled with aqueous KCl solutions (0.01, 0.1, and 1 M) [18b]. The value obtained for a 3.5/am-radius pipette was within the range from 10 to 10 As expected, the tip resistance was inversely proportional to the concentration of KCl in the filling solution. In ref. 18b, the effect of pipette radius on the tip resistance was evaluated using a constant concentration of KCl. The pipette resistance varied inversely with the tip radius. The iR drop was found to be 4.5-8 mV for the pipette radii of 0.6 to 19/rm when 10 mM KCl was used. 

FIG. 2. Simultaneous recording of membrane currents and Ca2+ fluorescence. (A) Upper and lower traces indicate the time courses of membrane current and [Ca2+] respectively. Cells were voltage-clamped at — 60 mV. Pipette contained Cs aspartate internal solution supplemented with 50 M fura-2. (B) Expanded time-courses of membrane current and [Ca2+] form the dotted box in A. TG, thapsigargin 2-APB, 2-aminoethoxydiphenyl borate. 

Erythrocyte suspensions are frozen at -20°C overnight prior to analysis. Pipette 100 pi of the internal standard working solution in a 4-ml glass vial. Add 50 pi of plasma or erythrocyte suspension. Add 1 ml of 3N methanolic HC1, close the vial with a screw cap and Teflon insert and allow the transmethylation to proceed at 90°C for 4 h. 

Though human error is usually the cause, proper method development can make a difference in reducing this type of error or variations. First, a large volume of IS, such as 200 pL should be used when it is added by a repeater pipette because small volumes (such as 50 pL or less) are more prone to imprecision than large ones. In addition, it would be extremely difficult to visually spot missed or doubled addition for an internal standard when its volume is much smaller than that of samples and/ or other reagents (e.g., buffer). Second, it would be helpful to reduce errors by adding the usually colorless IS solution first and then incurred samples, which are usually colored, such as slight yellowish for plasma samples or dark red for whole blood samples. 

The International Union of Pure and Applied Chemistry (IUPAC) recommends the use of liquid-liquid distribution over the traditional term solvent extraction.10 However, solvent extraction is still used prevalently in the literature. Solvent extraction utilizes the partition of a solute between two practically immiscible liquid phases one solvent phase and the other aqueous phase.11 A separatory funnel can be used in a lab to carry out solvent extraction. Of course, a simple test tube can also be used in conjunction with a glass pipette. The organic phase (solvent phase) is usually the top phase and the aqueous phase bottom phase. However, some organic solvents are heavier than water (for example, methylene chloride s specific gravity is 1.33 at 20°C) and in such cases the organic phase becomes the bottom phase. 

Procedure Packed erythrocytes, washed twice in Hepes buffer, are resuspended in an equal volume of the same buffer. The erythrocyte suspension (2 ml) is carefully layered over the Percoll-diatrizoate solution (2 ml) in a glass centrifuge tube with an internal diameter of 10 mm. After centrifugation at 400 x g for 20 min at room temperature, erythrocytes with normal density are carefully removed from the surface of the Percoll-diatrizoate cushion using a pasteur pipette. This fraction is referred to as the normal density fraction. Erythrocytes with elevated density, which had pelleted below the Percoll-diatrizoate, can then be removed in a similar manner. This latter fraction is referred to as the dense fraction. Finally, each fraction of erythrocytes is washed twice in 10 vol. of Hepes buffer at 4°C. 

Weigh in duplicate (to the nearest 0.1 mg) sufficient sample (vvg) to contain about 0.06 g of etofenprox into two 50-ml stoppered volumetric flasks. Into each flask pipette 10ml of di-cyclohexyl phthalate internal standard, shake the flasks thoroughly to dissolve the etofenprox and dilute to 50 ml with methanol/tetrahydrofuran (50 50, v/v) (solutions and Sb). 

IonWorks HT Load the reservoir in the buffer position with 4 ml PBS and cell position with cell suspension. Pipette the test compound, vehicle control, and positive control (threefold above the final concentration, DMSO <0.33 %) in a 96-well V-bottom plate. Load the plate in plate 1 position, and clamp PatchPlate into the PatchPlate station. With the fluidics-head (F-head) add 3.5 pi PBS to each well of PatchPlate and perfuse its underside with internal solution. Prime and de-bubble the electronics head (E-head), and perform hole test by applying voltage pulse. Dispense 3.5 pi of cell suspension in each well of PatchPlate with F-head. Allow 200 s for cells to reach the hole in each well, and seal it. Determine the seal resistance in each well with E-head. Change the underside solution of PatchPlate with access solution. After 9 min (perforation time) perform pre-compound hERG current measurements with E-head. Add 3.5 pi solution from each well of compound plate to 4 wells on a PatchPlate . 

It is probably more popular to perform microcalorimetry in the static mode. In the so-called internal hygrostat method, the compound under investigation is sealed into a vial with a sealed pipette tip containing the saturated salt solution chosen to give the required RH. 

The residue is dissolved in 2 ml of redistilled tetrahydrofuran (THF) containing 10 mg per 100 ml of coprostan-3 -ol (COP) as internal standard. (Blowout pipettes must not be used because moisture is detrimental to the formation of trimethyl silyl ethers.) The solution is transferred by capillary pipette to a 15-ml conical centrifuge tube, and the flask is rinsed out with 0.5 ml of fresh THF. Hexamethyldisilazane (0.3 ml) and trimethylchlorosilane (0.1 ml) are added. The tube is then stoppered, thoroughly mixed, and allowed to stand at room temperature overnight. 

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